Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exposure of MOLT-3 human leukemic cells in culture to a lipophilic antifolate, trimetrexate (TMQ), resulted in the development of sublines resistant to antifolates as well as to drugs related to multidrug resistance. The TMQ-resistant sublines had an increase in dihydrofolate reductase (DHFR) activity and overexpression of P-glycoprotein. In these sublines, neither the DHFR gene nor the MDR1 gene were amplified. In these cells, DHFR transcripts were also not overexpressed but DHFR protein was increased, indicative of translational or post-translational control of DHFR activity. In contrast, MDR1 transcripts were found to be overexpressed, in parallel with P-glycoprotein production. Therefore, increases in P-glycoprotein appear controlled at the transcriptional level. These data support evidence that TMQ produced two phenotypic changes independently: the former probably from folate deficiency and the latter from the lipophilic nature of the compound.
 ...
PMID:Expression of dihydrofolate reductase and multidrug resistance genes in trimetrexate-resistant human leukemia cell lines. 809 75

Toxoplasma gondii RH was obtained in high yield from culture in RPMI medium on a line of Chinese hamster ovary cells lacking dihydrofolate reductase activity (ATCC 3952 dhfr-; American Type Culture Collection). Dihydrofolate reductase preparations from harvested organisms had specific activities of 22.9 +/- 2.1 nmol/min/mg. The 50% inhibitory concentrations against reference compounds were 0.014 microM for methotrexate, 0.24 microM for pyrimethamine, 2.7 microM for trimethoprim, and 0.010 microM for trimetrexate. The Km value for NADPH was 11 microM and followed Michaelis-Menten kinetics; the Km for dihydrofolate was ca. 11 microM, but substrate inhibition appeared to occur at high substrate concentrations. Dihydrofolate reductase from T. gondii was used to screen 130 compounds from the National Cancer Institute repository. Thirteen compounds were > 100-fold more potent than pyrimethamine toward T. gondii dihydrofolate reductase; six compounds with various potencies were 8 to 46 times as selective as pyrimethamine for the protozoal form of the enzyme over the mammalian form. Four trimetrexate analogs were more potent than trimetrexate, and two were significantly more selective. Representative compounds were also tested in a culture model of T. gondii employing uracil incorporation as an index of growth. One pyrimethamine analog was as effective as pyrimethamine in inhibiting T. gondii in culture (50% inhibitory concentration, 0.45 microM). Three other compounds were also effective at micromolar concentrations.
 ...
PMID:Identification of highly potent and selective inhibitors of Toxoplasma gondii dihydrofolate reductase. 823 5

Dihydrofolate reductase-minus mutants of Chinese hamster ovary cells were depleted of glutathione peroxidase by transcription of the transfected bovine cDNA in inverted orientation upstream from the cDNA for dihydrofolate reductase to engender a bicistronic mRNA. In a clone of cells selected for expression of dihydrofolate reductase by the ability to grow in nucleoside-free medium the activity of glutathione peroxidase was reduced to 20% of the activity in the untransfected parental line of cells (DG44). The cells depleted of glutathione peroxidase were more sensitive to the toxicities of paraquat and adriamycin than the untransfected parental cells from which they derived but not more sensitive to bleomycin, menadione, or phenazine methosulfate. That the mildly increased sensitivity to paraquat and adriamycin was the consequence of the diminished cellular content of glutathione peroxidase was confirmed by the increase in sensitivity of untransfected cells after treatment with buthionine sulfoximine, an agent which depletes cells of glutathione. These and other data strongly suggest that the enzymic action of glutathione peroxidase protects cells from the toxicity of paraquat and adriamycin. The toxin which these agents engender is likely to be hydrogen peroxide or another hydroperoxide upon which glutathione peroxidase acts.
 ...
PMID:Glutathione peroxidase protects cultured mammalian cells from the toxicity of adriamycin and paraquat. 837 99

A chimeric gene was constructed encoding the entire murine dihydrofolate reductase (DHFR) protein with a carboxyl-terminal extension encompassing amino acids 494-795 of the rat glucocorticoid receptor (GR). The chimeric DHFR/GR gene encoded a functional DHFR protein, as measured by the ability to transform DHFR-deficient Chinese hamster ovary (CHO) cells to a DHFR-positive phenotype. The DHFR/GR protein bound [3H]dexamethasone with a similar affinity as wild-type GR. Selection of stable CHO transformants in increasing concentrations of methotrexate resulted in increased expression of DHFR/GR. Addition of dexamethasone, a synthetic glucocorticoid agonist, decreased the activity of the chimeric protein, as measured by colony formation in selective medium, binding of fluoresceinated methotrexate, and direct enzymatic assay for DHFR. Addition of RU486, a glucocorticoid antagonist, antagonized the effect of dexamethasone. In the absence of dexamethasone, the chimeric protein was primarily localized to the cytoplasm. In the presence of dexamethasone or RU486, DHFR/GR translocated into the nucleus. However, RU486 did not decrease DHFR activity, distinguishing subcellular location from functional activity. These results demonstrate that glucocorticoids negatively affect the function of DHFR/GR.
 ...
PMID:Dexamethasone negatively regulates the activity of a chimeric dihydrofolate reductase/glucocorticoid receptor protein. 848 45

The subcellular distributions of folate and folate-synthesizing enzymes were investigated in pea leaves. It was observed that the mitochondrial folate pool (approximately 400 micron) represented approximately 50% of the total pool. Furthermore, all the enzymes involved in tetrahydrofolate polyglutamate synthesis were present in the mitochondria. In marked contrast, we failed to detect any significant activity of these enzymes in chloroplasts, cytosol, and nuclei. The presence of the tetrahydrofolate synthesis pathway in mitochondria is apparently a general feature in plants since potato tuber mitochondria also contained a high folate concentration (approximately 200 micron) and all the enzymes required for tetrahydrofolate polyglutamate synthesis. The specific activities of tetrahydrofolate-synthesizing enzymes were rather low (1.5-15 nmol h-1 mg-1 matrix protein), except for dihydrofolate reductase (180-500 nmol h-1 mg-1 matrix protein). Dihydrofolate reductase was purified to homogeneity. The enzyme had a native molecular mass of approximately 140 kDa and was constituted of two identical 62-kDa subunits. Interestingly, this mitochondrial protein appeared to be a bifunctional enzyme, also supporting thymidylate synthesis. The cell distribution of thymidylate synthase was also investigated. No significant activity was observed in cell fractions other than mitochondria, indicating that plant cell mitochondria are also a major site for thymidylate synthesis.
 ...
PMID:Mitochondria are a major site for folate and thymidylate synthesis in plants. 862 17

Dihydrofolate reductase fusion proteins have been widely used to study conformational properties of polypeptides translocated across membranes. We have studied the import of dihydrofolate reductase fusion proteins into glycosomes and mitochondria of Trypanosoma brucei. As signal sequences we used the last 22 carboxy-terminal amino acids of glycosomal phosphoglycerate kinase for glycosomes, and the cleavable presequences of yeast cytochrome b2 or cytochrome oxidase subunit IV for mitochondria. Upon addition of aminopterin, a folate analogue that stabilizes the dihydrofolate reductase moiety, import of the fusion protein targeted to glycosomes was not inhibited, although the results of protease protection assays showed that the fusion protein could bind the drug. Under the same conditions, import of a DHFR fusion protein targeted to mitochondria was inhibited by aminopterin. When DHFR fusion proteins targeted simultaneously to both glycosomes and mitochondria were expressed, import into mitochondria was inhibited by aminopterin, whereas uptake of the same proteins into glycosomes was either unaffected or slightly increased. These findings suggest that the glycosomes possess either a strong unfolding activity or an unusually large or flexible translocation channel.
 ...
PMID:Import of a DHFR hybrid protein into glycosomes in vivo is not inhibited by the folate-analogue aminopterin. 863 10

In an attempt to introduce a large peptide that is not normally translocated across membranes into the cytosol of eukaryotic cells, we created a new chimeric protein termed CEDH between Pseudomonas aeruginosa exotoxin A (ETA) and a variant enzyme of Mus musculus dihydrofolate reductase (DHFR) with reduced affinity for antifolates, ETA(1-413).DHFR(1-187).ETA(609-613). We have defined, genetically constructed and expressed the chimeric protein in Escherichia coli. We showed that the CEDH chimeric protein, purified to homogeneity on an immunoaffinity resin, confers a methotrexate-resistant phenotype to Chinese hamster ovary cells. Furthermore, the chimeric protein allowed the growth of dihydrofolate reductase-deficient Chinese hamster ovary cells in the absence of hypoxanthine and thymidine. These results demonstrated that the chimeric protein exhibited enzyme activity and possessed the tightly folded native structure, and that the DHFR protein can be selectively internalized and translocated via domains of exotoxin A. These data show that the ETA system is an efficient system for the delivery of a variety of large polypeptides into the cytosol without stress to the target cells, and extends the use of this delivery system to proteins that are not normally translocated across membranes.
 ...
PMID:Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system. 884 33

Methotrexate (MTX) is a clinically important antifolate that has been used in combination with other chemotherapeutic agents in the treatment of malignancies including acute lymphocytic leukemia, osteosarcoma, carcinomas of the breast, head and neck, choriocarcinoma and non-Hodgkin's lymphoma. The primary target of MTX is the enzyme dihydrofolate reductase (DHFR) which catalyzes the reduction of folate and 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate. Understanding of MTX action has revealed how cells acquire resistance to this drug. The four known mechanisms of MTX resistance are a decrease in the uptake of the drug, a decrease in the retention of the drug due to defective polyglutamylation or an increase in polyglutamate breakdown, an increase in the enzyme activity and a decrease in the binding of MTX to DHFR. The molecular basis for some of these mechanisms has been elucidated in MTX resistant cell lines; in particular the occurrence of gene amplification resulting in increased DHFR and point mutations resulting in altered DHFR with reduced affinity for MTX. Cloning of the human folylpolyglutamate synthase gene and the reduced folate transport gene have been reported recently and should facilitate the identification of the molecular basis of these resistant phenotypes. DHFR protein has been shown to regulate its synthesis by exerting an inhibitory influence on its own translation. Addition of MTX relieves this inhibition thus providing a possible molecular explanation for the rapid rise in DHFR activity noted in some cells after MTX administration. Alterations in genes involved in regulating the cell cycle such as cyclin D1 and the retinoblastoma (Rb) gene have also been shown to influence cellular response to MTX. Overexpression of cyclin D1 in HT1080, a human fibrosarcoma cell line, results in decreased MTX sensitivity. The molecular basis of this observation is under investigation. Abnormalities in the Rb gene may also have profound effects on MTX sensitivity. Rb interacts with the family of transcription factors called E2F reducing transcription of genes that contain E2F binding sites in the promoter regions e.g. DHFR. When Rb is deleted or rendered nonfunctional levels of "free" or unbound E2F are high resulting in enhanced transcription of genes such as DHFR. This results in increased DHFR protein and may lead to MTX resistance. As the knowledge regarding mechanisms of resistance increases newer approaches to circumvent such resistance or to target resistant cells can be undertaken.
 ...
PMID:Molecular mechanisms of resistance to antifolates, a review. 885 36

Numerous new antifolate drugs have been developed in an attempt to overcome the potential mechanisms of tumor cell resistance to methotrexate, which can include decreased drug transport into cells; decreased polyglutamation, leading to increased drug efflux from cells; decreased drug affinity for folate-dependent enzymes; mutations of dihydrofolate reductase (DHFR), a key enzyme required for the maintenance of adequate intracellular reduced folate levels that is inhibited by methotrexate; and increased expression of the DHFR protein. Promising antifolate compounds undergoing clinical testing as anticancer agents include trimetrexate (which was recently approved by the FDA for the treatment of Pneumocystis carinii pneumonia), edatrexate, piritrexim, Tomudex, and lometrexol. The mechanisms of action, dosage, pharmacokinetics, clinical toxicity, and antitumor activity of these drugs are profiled.
 ...
PMID:New antifolates in clinical development. 892 75

Two small fragments of a novel human gammaherpesvirus genome known as Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV-8) have been shown to be present in virtually all AIDS and non-AIDS KS lesions, as well as in body cavity-based lymphomas (BCBL) and in multicentric Castleman's disease. We have extended those studies by identifying and sequencing a third fragment of HHV-8 DNA encoding a viral thymidylate synthetase (TS) gene. Use of this viral TS fragment as a probe led to the identification and mapping of a cluster of overlapping phage lambda clones from a BCBL tumor DNA genomic library that spanned 48 kb on the left-hand side of the HHV-8 genome between the equivalents of open reading frame 6 (ORF6) and ORF31 of herpesvirus saimiri (HVS). DNA sequencing of a 17-kb segment encompassing a gammaherpesvirus divergent locus (DL-B) between ORF11 and ORF17 revealed the presence of nine viral ORFs with predicted gene products related to cellular proteins. These include the complete TS gene and a dihydrofolate reductase (DHFR) gene, four novel cytokine genes (encoding viral interleukin-6, viral MIP-1A, viral MIP-1B, and BCK) that have not previously been found to be encoded by a virus, and a bcl-2 homolog. This region in HHV-8 also contains the T1.1 abundant lytic cycle nuclear RNA gene and encompasses two genes (or exons) encoding proteins with C4HC3 zinc finger domains of the PHD/leukemia-associated protein subtype. The latter are related to the spliced immediate-early IE1 protein of the gamma-2 class herpesvirus bovine herpesvirus type 4 and a similar motif found in HVS ORF12. Although genes for TS and DHFR enzymes are also encoded by HVS (ORF70 and ORF2), both occur at different genomic loci than in HHV-8, and the HHV-8 DHFR protein is much farther diverged from human DHFR than is the HVS version, implying that they were probably acquired as host cell cDNAs by independent evolutionary events. Transcripts from the IE1-A, IE1-B, DHFR, and MIP-1B genes were all detected by Northern blot hybridization analysis in a BCBL cell line at 12 h after induction with butyrate but were not present before induction, indicating that these are all primarily lytic cycle genes. We conclude that the DL-B locus of gammaherpesviruses displays considerably more variability that previously appreciated and that expression of many of these genes is likely to have important implications for HHV-8 biology and therapy.
 ...
PMID:A single 13-kilobase divergent locus in the Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genome contains nine open reading frames that are homologous to or related to cellular proteins. 903 28


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>